High-resolution, scanning fluorescence microscopy methods, such as confocal microscopy or multi-photon microscopy, using standard refractive optics face the limitation of a trade-off between resolution and signal detection sensitivity on the one hand versus the image area (field of view) of the acquired image on the other.
Applications in the field of life sciences, histology and clinical tissue imaging using high-resolution fluorescence microscopy would largely benefit from overcoming this trade-off.
Imaging large surfaces of cell cultures, ex-vivo tissue samples and in-vivo tissue structure would enhance the efficiency of automatized imaging procedures in biology and histology and increase the quality of medical diagnosis based on histological imaging ex-vivo and in-vivo.
Overcoming the above mentioned trade-off and at the same time miniaturising the microscopy device would have a significant impact on endoscope based applications of microscopy imaging of internal organs in a non-invasive manner.
In the clinical environment, microscopic imaging by means of confocal scanning endomicroscopy inside the living patient is a recent trend in endoscopy. It has the potential to provide the physician with histological insights of the tissue, helping to discriminate healthy from potentially malignant or diseased tissue.
Confocal scanning endomicroscopy, similar to standard confocal microscopy, requires a fluorescent stain to produce image contrast by means of fluorescence excitation. Either endogenous autofluorescence or a fluorescent agent can be excited by means of laser light. Confocal scanning endomicroscopy has shown very promising results for cancer detection and tissue characterisation during on-going clinical procedures (Kiesslich et al, Atlas of Endomicroscopy, Springer 2008).
The two Endomicroscopy systems that are used in clinics today use fundamentally different technologies. Optiscan Imaging has developed a miniaturized MEMS based scanner that is integrated into the distal end of a specific endoscope, which is marketed as a complete system for gastroenterological endoscopy (Evans and Nishioka, Curr Opin Gastroenterol 2005; 21: 578-584). The Optiscan system uses small lenses to focus the light into the tissue of interest, which consequently results in a field of view which is significantly smaller than the device dimension.
On the other hand, Mauna Kea Technologies has developed a multi-core fiber based system that is scanned on the proximal end of the fiber bundle, where each fiber core serves as a pinhole (Thiberville et al., Proc Am Thorac Soc 2009, 6: 444-449). The imaging fiber is either used with a focusing lens at the distal tip or without any focusing optics. If no focusing optics is used, the ratio of device size to field of view can be increased at the expense of image resolution.
The size of the focusing optics has a significant impact on the application fields of endoscopy. For instance, Mauna Kea Technologies' system is a thin probe that can be inserted through the working channel of standard endoscopes, and thus be applied in different medical fields, such as gastroenterology, pulmonology or urology.
In this context, the present invention advantageously addresses the following points:                Keeping high resolution while increasing the field of view;        Improving the ratio of device dimension to field of view, close to one;        Confocal imaging; and        Increase of imaging depth in tissue.        
Imaging large tissue areas with high resolution, while taking up less space in the tip of the endoscope, significantly increases the medical utility of endomicroscopy for performing live-histology (optical biopsy) of living tissue.
Increasing the imaging depth in order to reach the sub-mucosal layer during an endomicroscopy procedure would open the possibility to perform accurate staging of early-stage tumours. Precise tumour staging is key in appropriate therapy choice and influences largely patient mortality and morbidity as well as the overall cost of the treatment.
Endomicroscopy is a novel medical field (Waldner et al, Nat Protoc 2011; 6(9): 1471-81, 2011 and citations therein), dating back to the early 2000's. Integrating a miniaturized fluorescence microscope into a medical endoscope provides the endoscopist with the possibility to see the cellular structure of the tissue under investigation, similar to conventional histology after biopsy extraction. Using confocal endomicroscopes and appropriate fluorescent dyes allows for image sectioning in the axial direction. This insight into the deeper layers of the tissue during ongoing procedures is important for a precise medical diagnosis of tissue disorders, such as early-stage tumors or pre-tumorous conditions (dysplasia f.i.).
A multitude of patents have been filed since the year 2000, either to protect the method of confocal endomicroscopy, the particular technological implementation of endomicroscopes or integration aspects into endoscopes, by the companies principally active in commercializing Endomicroscopy systems: Optiscan Imaging (Dabbs et al. 1990, WO9001716) in Marketing collaboration with Pentax Medical, and Mauna Kea Technologies (Viellerobe et al. 2001, WO2003056378).
In recent years, a new wave of prototypes for the next generation confocal scanning endomicroscopes (Jabbour et al., Ann Biomed Eng 2012; 40(2): 378-97, 2012 and citations therein) have been developed and patented or published. In parallel, much work has been done by different groups to develop non-linear scanning endomicroscopes (Wu & Li, Handbook of Photonics for Biomedical Science (Edited by V. V. Tuchin), CRC Press 2010, 547-74 and citations therein; Ben-Yakar et al. 2011, WO2011091283). Non-linear fluorescence imaging is technologically more sophisticated to implement into an endoscope, but offers the following advantages over confocal fluorescence imaging for in-vivo medical applications (Helmchen and Denk, Nat Methods 2005, 2(12): 932-40, 2005):                Inherent confocal sectioning due to non-linear fluorescence excitation;        Better collection of excited fluorescence, due to avoiding a pinhole;        Imaging deeper in the tissue, if near-infrared lasers are used for fluorescence excitation;        Possibility to excite second harmonic generation and image without external dyes inherent tissue features, such as collagen fibers; and        Less overall photo-damage at similar laser powers, due to fluorescence excitation confined to the focal spot.        
The great majority of the published or patented endomicroscopy devices use small, single refractive elements for light focusing in the tissue of interest. Such refractive elements are typically small lenses or gradient-index lenses (GRIN). These elements suffer from stronger optical aberrations compared to standard, corrected microscope objectives. Furthermore, these elements are limited in the numerical aperture that they can reach. The numerical aperture defines the resolution of the image, the fluorescence collection efficiency and the efficiency of fluorescence excitation.
Webb and Xu proposed in 2009 (Webb et al. 2009, WO2009064746) a focusing system for a non-linear microscopy endoscope with two distinct regimes for the focusing of excitation light and the collection of fluorescence light. Using a dichroic layer on a lens—adapted to transmit the spectral range of the fluorescence light and to reflect the spectral range of the excitation light—the excitation light is focused on the basis of light reflection and the fluorescence light is collected on the basis of refraction with the same coated high numerical aperture lens. The advantage of Webb's and Xu's approach is the increased fluorescence excitation efficiency, due to excitation light focusing by means of a reflector. The arrangement of the reflective surfaces is very similar to the well-known design of a Schwarzschild microscope objective. Webb and Xu propose the usage of only one, macroscopic, such Schwarzschild-type element, resulting in a ratio between the image field-of-view and the overall device cross-section dimensions, far from the ratio of 1:1, and comparable to ratios of purely refractive systems.
In spite of a range of clinical benefits, current endomicroscopy systems are criticized by its end-users, the physicians, for several aspects. The subjects of the most recurrent criticisms are: increased overall procedure duration, long learning curve or the high price of equipment.
From the technological point of view, improvements of the image acquisition rate (reduction of motion artifacts), imaging of deeper tissue sections for better tumor staging or a larger image field of view while maintaining high contrast and resolution are requested, as summarized by Hwang (Hwang 2009) for applications the upper GI for instance.
The trade-off between the image area and image resolution/definition—is a known and barely resolved problem in microscopy. If a large image with high resolution is required, the obvious solution is to sequentially image small sample areas with high resolution. Reconstructing the image with mosaic stitching algorithms is the subsequent step.
However, this is highly time consuming and can additionally result in image artefacts. If scanning microscopy techniques such as confocal microscopy or multiphoton microscopy are considered, then the time consuming aspect becomes even more striking, since the scanning image acquisition is several factors slower than wide-field imaging.
To improve the performance of refractive elements, such as lenses, to stretch the above mentioned trade-off while remaining in typical scanning time frames, the most recent and significant was done by Amos et al. (Saini, Science 2012, 335(6076): 1562-3) who proposed a “mesolens”.
This mesolens is a large lens that allows for confocal imaging of an entire embryo while maintaining resolution of single cells. However, in the case of the “mesolens” the overall dimensions of the microscope increase to several tens of centimeters, making it unsuitable for endomicroscopic applications. Optical aberrations have to be corrected with additional optical elements, which is valid for most optical systems that use refractive elements. Furthermore, using even such sophisticated lens systems as the mesolens, the maximal achieved image size remains in the order of a few millimeters.
In 2009 Rachet et al. (Rachet et al. 2009, WO2010084478) proposed a solution for overcoming this trade-off using an array of focusing micromirrors within a confocal scanning laser fluorescence microscope.
However, the microscopy device of WO2010084478 requires a thin ex-vivo sample preparation and is not suited for epi-fluorescence, forward-imaging of thick tissue in-vivo or living tissue of a patient in a clinical environment. Moreover, the collected light has to traverse the thin sample before the signal is detected by a camera or sensor.
Reflective microscope objectives are also known, however, these objectives are bulky and unsuitable for in-vivo imaging. Moreover, these objectives require a supporting structure for their optical reflectors that results in a loss of the optical signal transferred through the objective.
A goal of the present invention is to solve the above mentioned problems and in particular to provide an optical element permitting high resolution imaging of a large image area, increased imaging depth in tissue, that can be miniaturised for parallel imaging of large areas with high resolution and that also permits imaging of thick tissue in-vivo or living tissue of a patient in a clinical environment.